Project Summary Embryonic development and tissue homeostasis rely on the tight regulation of cell-type specific transcriptional programs by particular transcription factors (TFs) and their target enhancers. The emergence of three- dimensional (3D) genome organization as an important layer of transcriptional control, stresses the fact that understanding how enhancers communicate with target genes to coordinate transcriptional activity requires knowledge of the 3D nuclear topology. In a recent study, we captured a drastic rewiring of three-dimensional regulatory contacts between somatic cells and embryonic stem cells (ESCs) by H3K27ac HiChIP and identified complex 3D ?enhancer hubs?, where enhancers are spatially clustered with multiple highly-expressed genes with known or predicted functions in regulation of stemness. Genetic or epigenetic modulation of such enhancers in ESCs resulted in downregulation of all hub-connected genes and partial differentiation, which supports a vital role for these architectural nodes in gene coregulation and cell identity. In addition, we provided proteomics and genetic evidence that KLF family TFs play an important role in the organization and regulation of 3D enhancer hubs in ESCs and identified candidate cofactors. Based on these results, we hypothesize that 3D enhancer hubs function as architectural ?headquarters? of cell identity, where cell type-specific genes are sequestered by specific transcriptional regulators to facilitate coordinated gene expression. Here, we will test this hypothesis both in the contexts of mouse ESCs, and of early developmental cell fate decisions using in vitro and in vivo approaches. Specifically, we aim to (1) target systematically enhancers and genes within hubs to determine the functional consequences on the pluripotent transcriptional network and the stability of ESC identity, (2) determine the critical protein factors and activities that control enhancer hub formation and functionality and (3) identify and characterize 3D enhancer hubs that are critical for acquisition and maintenance of each of the early developmental fates. Successful completion of our aims will offer mechanistic insights into the organization and regulation of 3D enhancer hubs, determine their role in cell fate control and reveal novel ways for engineering cell identity by targeting critical architectural nodes and factors.